1. Field of the Invention
The invention relates to the field of plant biotechnology. More particularly, the invention relates to identification and use of chloroplast transit peptides allowing efficient processing and localization of dicamba monooxygenase enzymes in plants.
2. Description of the Related Art
DMO (dicamba monooxygenase) catalyzes the degradation of the herbicide dicamba (3,6-dichloro-o-anisic acid) to non-toxic 3,6-dichlorosalicylic acid (3,6-DCSA) in plants, thus conferring herbicide tolerance. Activity of DMO requires two intermediary proteins for shuttling electrons from NADH to dicamba, a reductase and a ferredoxin (U.S. Pat. No. 7,022,896; Herman et al., 2005). However dicamba tolerance in transgenic plants has been demonstrated through transformation with DMO alone, indicating that a plant's endogenous reductase and ferredoxin may substitute in shuttling the electrons. The plant ferredoxin that is involved in electron transfer is localized in the plastids. Thus, in order to obtain efficient performance of DMO and thus improved tolerance to dicamba, there is a need for targeting the DMO to chloroplasts.
In many cases, this targeting may be achieved by the presence of an N-terminal extension, called a chloroplast transit peptide (CTP) or plastid transit peptide. Chromosomal transgenes from bacterial sources must have a sequence encoding a CTP sequence fused to a sequence encoding an expressed polypeptide if the expressed polypeptide is to be compartmentalized in the plant plastid (e.g. chloroplast). Accordingly, localization of an exogenous polypeptide to a chloroplast is often accomplished by means of operably linking a polynucleotide sequence encoding a CTP sequence to the 5′ region of a polynucleotide encoding the exogenous polypeptide. The CTP is removed in a processing step during translocation into the plastid. Processing efficiency may, however, be affected by the amino acid sequence of the CTP and nearby sequences at the NH2 terminus of the peptide.
Weeks et al. (U.S. Pat. No. 7,022,896) describe potential use of a maize cab-m7 signal sequence (see Becker et al., 1992 and PCT WO 97/41228; GenBank Accession No. X53398) and a pea glutathione reductase signal sequence (Creissen et al., 1992 and PCT WO 97/41228) in targeting DMO to plant plastids, but no data on efficiency of processing or targeting is given. A pea Rubisco small subunit (RbcS) CTP including a 27 aa sequence including coding sequence for pea Rubisco enzyme small subunit has also been used to target DMO to chloroplasts (e.g. U.S. Provisional Application Ser. No. 60/811,152). However, it has been found during Western blot analysis that this pea RbcS CTP generates a correctly processed DMO protein band (˜38 kDa), but also a larger band (˜41 kDa) corresponding to that of DMO and the 27 aa of RbcS coding region. The extra amino acids could impact the DMO activity adversely. In addition, additional proteins in a transgenic product due to incomplete processing of DMO create regulatory hurdles and require additional efforts in characterization of the product for the purposes of product registration by government agencies thereby raising the cost of product registration. Thus, there is a need for identifying CTPs that efficiently generate correctly processed DMO, thereby providing the advantage of full DMO activity as well as ease of product characterization.